The invention relates to nucleic acid detection. More specifically, the invention relates to the detection of targeted, predetermined endogenous nucleic acid sequences in nucleic acid target hybrids, and the various applications of their detection.
Methods to detect nucleic acids and to detect specific nucleic acids of interest provide a foundation upon which the large and rapidly growing field of molecular biology is built. There is constant need for alternative methods and products. The reasons for selecting one method over another are varied, and include a desire to avoid radioactive materials, the lack of a license to use a technique, the cost or availability of reagents or equipment, the desire to minimize the time spent or the number of steps, the accuracy or sensitivity for a certain application, the ease of analysis, or the ability to automate the process.
The detection of nucleic acids or specific nucleic acids is often a portion of a process rather than an end in itself. There are many applications of the detection of nucleic acids in the art, and new applications are always being developed. The ability to detect and quantify nucleic acids is useful in detecting microorganisms, viruses and biological molecules, and thus affects many fields, including human and veterinary medicine, food processing and environmental testing. Additionally, the detection and/or quantification of specific biomolecules from biological samples (e.g. tissue, sputum, urine, blood, semen, saliva) has applications in forensic science, such as the identification and exclusion of criminal suspects and paternity testing as well as medical diagnostics.
Some general methods to detect nucleic acids are not dependent upon a priori knowledge of the nucleic acid sequence. A nucleic acid detection method that is not sequence specific, but is RNA specific is described in U.S. Pat. No. 4,735,897, where RNA is depolymerized using a polynucleotide phosphorylase (PNP) in the presence of phosphate or using a ribonuclease. PNP stops depolymerizing when a double-stranded RNA segment is encountered, sometimes as the form of secondary structure of single-stranded RNA, as is common in ribosomal RNA, transfer RNA, viral RNA, and the message portion of mRNA. PNP depolymerization of the polyadenylated tail of mRNA in the presence of inorganic phosphate forms ADP. Alternatively, depolymerization using a ribonuclease forms AMP. The formed AMP is converted to ADP with myokinase, and ADP is converted into ATP by pyruvate kinase or creatine phosphokinase. Either the ATP or the byproduct from the organophosphate co-reactant (pyruvate or creatine) is detected as an indirect method of detecting mRNA.
In U.S. Pat. No. 4,735,897, ATP is detected by a luciferase detection system. In the presence of ATP and oxygen, luciferase catalyzes the oxidation of luciferin, producing light that can then be quantified using a luminometer. Additional products of the reaction are AMP, pyrophosphate and oxyluciferin.
Duplex DNA can be detected using intercalating dyes such as ethidium bromide. Such dyes are also used to detect hybrid formation.
Hybridization methods to detect nucleic acids are dependent upon knowledge of the nucleic acid sequence. Many known nucleic acid detection techniques depend upon specific nucleic acid hybridization in which an oligonucleotide probe is hybridized or annealed to nucleic acid in the sample or on a blot, and the hybridized probes are detected.
A traditional type of process for the detection of hybridized nucleic acid uses labeled nucleic acid probes to hybridize to a nucleic acid sample. For example, in a Southern blot technique, a nucleic acid sample is separated in an agarose gel based on size and affixed to a membrane, denatured, and exposed to the labeled nucleic acid probe under hybridizing conditions. If the labeled nucleic acid probe forms a hybrid with the nucleic acid on the blot, the label is bound to the membrane. Probes used in Southern blots have been labeled with radioactivity, fluorescent dyes, digoxygenin, horseradish peroxidase, alkaline phosphatase and acridinium esters.
Another type of process for the detection of hybridized nucleic acid takes advantage of the polymerase chain reaction (PCR). The PCR process is well known in the art (U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159). To briefly summarize PCR, nucleic acid primers, complementary to opposite strands of a nucleic acid amplification target sequence, are permitted to anneal to the denatured sample. A DNA polymerase (typically heat stable) extends the DNA duplex from the hybridized primer. The process is repeated to amplify the nucleic acid target. If the nucleic acid primers do not hybridize to the sample, then there is no corresponding amplified PCR product. In this case, the PCR primer acts as a hybridization probe. PCR-based methods are of limited use for the detection of nucleic acid of unknown sequence.
In a PCR method, the amplified nucleic acid product may be detected in a number of ways, e.g. incorporation of a labeled nucleotide into the amplified strand by using labeled primers. Primers used in PCR have been labeled with radioactivity, fluorescent dyes, digoxygenin, horseradish peroxidase, alkaline phosphatase, acridinium esters, biotin and jack bean urease. PCR products made with unlabeled primers may be detected in other ways, such as electrophoretic gel separation followed by dye-based visualization.
Fluorescence techniques are also known for the detection of nucleic acid hybrids. U.S. Pat. No. 5,691,146 describes the use of fluorescent hybridization probes that are fluorescence-quenched unless they are hybridized to the target nucleic acid sequence. U.S. Pat. No. 5,723,591 describes fluorescent hybridization probes that are fluorescence-quenched until hybridized to the target nucleic acid sequence, or until the probe is digested. Such techniques provide information about hybridization, and are of varying degrees of usefulness for the determination of single base variances in sequences. Some fluorescence techniques involve digestion of a nucleic acid hybrid in a 5xe2x80x2xe2x86x923xe2x80x2 direction to release a fluorescent signal from proximity to a fluorescence quencher, for example, TaqMan(copyright) (Perkin Elmer; U.S. Pat. Nos. 5,691,146 and 5,876,930).
Enzymes having template-specific polymerase activity for which some 3xe2x80x2xe2x86x925xe2x80x2 depolymerization activity has been reported include E. coli DNA Polymerase (Deutscher and Kornberg, J. Biol. Chem., 244(11):3019-28 (1969)), T7 DNA Polymerase (Wong et al., Biochemistry 30:526-37 (1991); Tabor and Richardson, J. Biol. Chem. 265:8322-28 (1990)), E. coli RNA polymerase (Rozovskaya et al., Biochem. J. 224:645-50 (1994)), AMV and RLV reverse transcriptases (Srivastava and Modak, J. Biol. Chem. 255:2000-4 (1980)), and HIV reverse transcriptase (Zinnen et al., J. Biol. Chem. 269:24195-202 (1994)). A template-dependent polymerase for which 3xe2x80x2 to 5xe2x80x2 exonuclease activity has been reported on a mismatched end of a DNA hybrid is phage 29 DNA polymerase (de Vega, M. et al. EMBO J., 15:1182-1192, 1996).
A variety of methodologies currently exist for detection of single nucleotide polymorphisms (SNPs) that are present in genomic DNA. SNPs are DNA point mutations or insertions/deletions that are present at measurable frequencies in the population. SNPs are the most common variations in the genome. SNPs occur at defined positions within genomes and can be used for gene mapping, defining population structure, and performing functional studies. SNPs are useful as markers because many known genetic diseases are caused by point mutations and insertions/deletions.
In rare cases where an SNP alters a fortuitous restriction enzyme recognition sequence, differential sensitivity of the amplified DNA to cleavage can be used for SNP detection. This technique requires that an appropriate restriction enzyme site be present or introduced in the appropriate sequence context for differential recognition by the restriction endonuclease. After amplification, the products are cleaved by the appropriate restriction endonuclease and products are analyzed by gel electrophoresis and subsequent staining. The throughput of analysis by this technique is limited because samples require processing, gel analysis, and significant interpretation of data before SNPs can be accurately determined.
Single strand conformational polymorphism (SSCP) is a second technique that can detect SNPs present in an amplified DNA segment (Hayashi, K. Genetic Analysis: Techniques and Applications 9:73-79, 1992). In this method, the double stranded amplified product is denatured and then both strands are allowed to reanneal during electrophoresis in non-denaturing polyacrylamide gels. The separated strands assume a specific folded conformation based on intramolecular base pairing. The electrophoretic properties of each strand are dependent on the folded conformation. The presence of single nucleotide changes in the sequence can cause a detectable change in the conformation and electrophoretic migration of an amplified sample relative to wild type samples, allowing SNPs to be identified. In addition to the limited throughput possible by gel-based techniques, the design and interpretation of SSCP based experiments can be difficult. Multiplex analysis of several samples in the same SSCP reaction is extremely challenging. The sensitivity required in mutation detection and analysis has led most investigators to use radioactively labeled PCR products for this technique.
In the amplification refractory mutation system (ARMS, also known as allele specific PCR or ASPCR), two amplification reactions are used to determine if a SNP is present in a DNA sample (Newton et al. Nucl Acids Res 17:2503, 1989; Wu et al. PNAS 86:2757, 1989). Both amplification reactions contain a common primer for the target of interest. The first reaction contains a second primer specific for the wild type product which will give rise to a PCR product if the wild type gene is present in the sample. The second PCR reaction contains a primer that has a single nucleotide change at or near the 3xe2x80x2 end that represents the base change that is present in the mutated form of the DNA. The second primer, in conjunction with the common primer, will only function in PCR if genomic DNA that contains the mutated form of genomic DNA is present. This technique requires duplicate amplification reactions to be performed and analyzed by gel electrophoresis to ascertain if a mutated form of a gene is present. In addition, the data must be manually interpreted.
Single base extension is a technique that allows the detection of SNPs by hybridizing a single strand DNA probe to a captured DNA target (Nikiforov, T. et al. Nucl Acids Res 22:4167-4175). Once hybridized, the single strand probe is extended by a single base with labeled dideoxynucleotides. The labeled, extended products are then detected using calorimetric or fluorescent methodologies.
A variety of technologies related to real-time (or kinetic) PCR have been adapted to perform SNP detection. Many of these systems are platform based, and require specialized equipment, complicated primer design, and expensive supporting materials for SNP detection. In contrast, the process of this invention has been designed as a modular technology that can use a variety of instruments that are suited to the throughput needs of the end-user. In addition, the coupling of luciferase detection sensitivity with standard oligonucleotide chemistry and well-established enzymology provides a flexible and open system architecture. Alternative analytical detection methods, such as mass spectroscopy, HPLC, and fluorescence detection methods can also be used in the process of this invention, providing additional assay flexibility.
SNP detection using real-time amplification relies on the ability to detect amplified segments of nucleic acid as they are during the amplification reaction. Three basic real-time SNP detection methodologies exist: (i) increased fluorescence of double strand DNA specific dye binding, (ii) decreased quenching of fluorescence during amplification, and (iii) increased fluorescence energy transfer during amplification (Wittwer, C. et al. Biotechniques 22:130-138, 1997). All of these techniques are non-gel based and each strategy will be briefly discussed.
A variety of dyes are known to exhibit increased fluorescence in response to binding double stranded DNA. This property is utilized in conjunction with the amplification refractory mutation system described above to detect the presence of SNP. Production of wild type or mutation containing PCR products are continuously monitored by the increased fluorescence of dyes such as ethidium bromide or SYBER Green as they bind to the accumulating PCR product. Note that dye binding is not selective for the sequence of the PCR product, and high non-specific background can give rise to false signals with this technique.
A second SNP detection technology for real time PCR, known generally as exonuclease primers (TaqMan(copyright) probes), utilizes the 5xe2x80x2 exonuclease activity of thermostable polymerases such as Taq to cleave dual-labeled probes present in the amplification reaction (Wittwer, C. et al. Biotechniques 22:130-138, 1997; Holland, P et al PNAS 88:7276-7280, 1991). While complementary to the PCR product, the probes used in this assay are distinct from the PCR primer and are dually-labeled with both a molecule capable of fluorescence and a molecule capable of quenching fluorescence. When the probes are intact, intramolecular quenching of the fluorescent signal within the DNA probe leads to little signal. When the fluorescent molecule is liberated by the exonuclease activity of Taq during amplification, the quenching is greatly reduced leading to increased fluorescent signal.
An additional form of real-time PCR also capitalizes on the intramolecular quenching of a fluorescent molecule by use of a tethered quenching moiety. The molecular beacon technology utilizes hairpin-shaped molecules with an internally-quenched fluorophore whose fluorescence is restored by binding to a DNA target of interest (Kramer, R. et al. Nat. Biotechnol. 14:303-308, 1996). Increased binding of the molecular beacon probe to the accumulating PCR product can be used to specifically detect SNPs present in genomic DNA.
A final general fluorescent detection strategy used for detection of SNPs in real time utilizes synthetic DNA segments known as hybridization probes in conjunction with a process known as fluorescence resonance energy transfer (FRET) (Wittwer, C. et al. Biotechniques 22:130-138, 1997; Bernard, P. et al. Am. J. Pathol. 153:1055-1061, 1998). This technique relies on the independent binding of labeled DNA probes on the target sequence. The close approximation of the two probes on the target sequence increases resonance energy transfer from one probe to the other, leading to a unique fluorescence signal. Mismatches caused by SNPs that disrupt the binding of either of the probes can be used to detect mutant sequences present in a DNA sample.
A number of gene-level defects have been implicated in the etiology of human disease. Researchers have used several techniques to detect these genetic mutations for prevention or diagnosis of these disease states. Van Essen et al. [J. Med. Genet. 34:805-12 (1997)] report that 65-70% of Duchenne and Becker muscular dystrophy patients exhibit rearrangements in the dystrophin gene, as detected by Southern blotting or multiplex PCR. Microlesions in these two forms of muscular dystrophy are typically detected using single strand conformational analysis, heteroduplex analysis, and the protein truncation test.
Calvano et al. [Clin. Genet. 52:17-22 (1997)] report the use of PCR fragments used as fluorescent probes for the detection of female carriers of Duchenne and Becker muscular dystrophy. Jongpiputvanich et al. [J. Med. Assoc. Thai. 79(Supp. 1):S15-21 (1996)] report the use of multiplex PCR and microsatellite or STR analysis for diagnosis and carrier detection in a Duchenne muscular dystrophy family. Pastore et al. [Mol. Cell. Probes 10:129-37 (1996)] developed a quantitative PCR analysis method using radiolabeled PCR products for the detection of macrodeletion carriers of Duchenne and Becker muscular dystrophy. Katayama et al. [Fetal Diagn. Ther. 9:379-84 (1994)] studied the efficacy of PCR for prenatal diagnosis of Duchenne muscular dystrophy. These workers used PCR-restriction fragment length polymorphism analysis, multiplex PCR, and dinucleotide repeat polymorphism analysis to diagnose affected male fetuses and detect carrier female fetuses in the first trimester.
A polymorphism in the human gap junctional protein connexin 37 was studied as a prognostic marker for atherosclerosis. Boerma et al. Intern. Med. 246:211-218 (1999). A restriction fragment length polymorphism in the proline variant of the connexin 37 gene was used to show that this allele was over-represented in patients with atherosclerotic plaques. Shohet et al. [Arterioscler. Thromb. Vasc. Biol. 19:1975-78 (1999)] report the frequency of the xe2x88x92514T allele of hepatic lipase in white men with coronary artery disease. In this population, postheparin plasma hepatic lipase activity was 15 to 20% lower in heterozygotes and 30% lower in homozygotes compared to controls. A novel missense mutation in the presenilin-1 gene was detected in a family with presenile familial Alzheimer""s disease (FAD). Sugiyama et al. Mutat. 14:90 (1999). These workers report that over 50 such missense mutations in the presenilin-1 gene have been reported in families with FAD. Sensitive, reliable assays for these and other gene-level defects have several potential diagnostic and preventative applications in human and animal health care.
In summary, there is a need for alternative methods for the detection of nucleic acid hybrids. There is a great demand for such methods to determine the presence or absence of nucleic acid sequences that differ slightly from sequences that might otherwise be present. There is a great demand for methods to determine the presence or absence of sequences unique to a particular species in a sample. There is also a great demand for methods that are more highly sensitive than the known methods, highly reproducible and automatable.
It would be beneficial if another method were available for detecting the presence of a sought-after, predetermined target nucleotide sequence or allelic variant. It would also be beneficial if such a method were operable using a sample size of the microgram to picogram scale. It would further be beneficial if such a detection method were capable of providing multiple analyses in a single assay (multiplex assays). The disclosure that follows provides such methods.
A method of this invention is used to determine the presence or absence of a predetermined (known) endogenous nucleic acid target sequence in a nucleic acid sample. Such a method utilizes an enzyme that can depolymerize the 3xe2x80x2-terminus of an oligonucleotide probe hybridized to a nucleic acid target sequence to release one or more identifier nucleotides whose presence can then be determined.
One embodiment of the invention contemplates a method for determining the presence or absence of a predetermined endogenous nucleic acid target sequence in a nucleic acid sample. Thus, the presence or absence of at least one predetermined endogenous nucleic acid target sequence is sought to be determined. More than one such predetermined endogenous target sequence can also be present in the sample being assayed, and the presence or absence of more than one predetermined endogenous nucleic acid target sequence can be determined. The embodiment comprises the following steps.
A treated sample is provided that may contain a predetermined endogenous nucleic acid target sequence hybridized with a nucleic acid probe that includes an identifier nucleotide in the 3xe2x80x2-terminal region. The treated sample is admixed with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3xe2x80x2-terminus of a hybridized nucleic acid probe to form a treated reaction mixture. The treated reaction mixture is maintained under depolymerizing conditions for a time period sufficient to permit the enzyme to depolymerize hybridized nucleic acid and release identifier nucleotides therefrom.
An analytical output is obtained by analyzing for the presence or absence of released identifier nucleotides. The analytical output indicates the presence or absence of the nucleotide at the predetermined region, and, thereby, the presence or absence of a first nucleic acid target. The analytical output is obtained by various techniques as discussed herein.
It is contemplated that an analytical output of the methods of the invention can be obtained in a variety of ways. The analytical output can be ascertained by luminescence spectroscopy. In some preferred embodiments, analysis for released 3xe2x80x2-terminal region indicator nucleotides comprises the detection of ATP, either by a luciferase detection system (luminescence spectroscopy) or an NADH detection system (absorbance spectroscopy). In particularly preferred embodiments where greater sensitivity is desired, ATP molecules are formed by a phosphate transferring step, for example using an enzyme such as NDPK in the presence of ADP, from the nucleoside triphosphates produced by the depolymerizing step. In some embodiments the ATP is amplified to form a plurality of ATP molecules. In the ATP detection embodiments, typically the enzyme (NDPK) for converting nucleotides and added ADP into ATP is present in the depolymerization reaction with the depolymerizing enzyme, and when they are present together, they are denoted as a xe2x80x9cone potxe2x80x9d method.
In an alternative embodiment, the analytical output is obtained by fluorescence spectroscopy. Use of a wide variety of fluorescence detection methods is contemplated. In one exemplary contemplated method, an identifier nucleotide includes a fluorescent label. An identifier nucleotide can be fluorescently labeled prior to, or after, release of the identifier nucleotide. It is also contemplated that other than a released identifier nucleotide contains a fluorescent tag. In such an embodiment, the release of nucleotides in a process of the invention is ascertained by a determination of a difference in the length of the polynucleotide probe, for example by capillary electrophoresis imaged by a fluorescent tag at the 5xe2x80x2 terminus of the probe or in a region other than the 3xe2x80x2 terminal region.
In an alternative embodiment the analytical output is obtained by mass spectrometry. It is preferred here that an identifier nucleotide be a nucleotide analog or a labeled nucleotide and have a molecular mass that is different from the mass of a usual form of that nucleotide, although a difference in mass is not required. It is also noted that with a fluorescently labeled identifier nucleotide, the analytical output can also be obtained by mass spectrometry. It is also contemplated that the analysis of released nucleotide be conducted by ascertaining the difference in mass of the probe after a depolymerization step of a process of the invention.
In another alternative embodiment, the analytical output is obtained by absorbance spectroscopy. Such analysis monitors the absorbance of light in the ultraviolet and visible regions of the spectrum to determine the presence of absorbing species. In one aspect of such a process, released nucleotides are separated from hybridized nucleic acid and other polynucleotides by chromatography (e.g. HPLC or GC) or electrophoresis (e.g. PAGE or capillary electrophoresis). Either the released identifier nucleotide or the remainder of the probe can be analyzed for to ascertain the release of the identifier nucleotide in a process of the invention. In another aspect of such a process a label may be incorporated in the analyzed nucleic acid.
In a contemplated embodiment, a sample to be assayed is admixed with one or more nucleic acid probes under hybridizing conditions to form a hybridization composition. The 3xe2x80x2-terminal region of the nucleic acid probe hybridizes with partial or total complementarity to the nucleic acid target sequence when that sequence is present in the sample. The 3xe2x80x2-terminal region of the nucleic acid probe includes an identifier nucleotide. The hybridization composition is maintained under hybridizing conditions for a time period sufficient to form a treated sample that may contain said predetermined nucleic acid target sequence hybridized with a nucleic acid probe. The treated sample is admixed with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3xe2x80x2-terminus of a hybridized nucleic acid probe to form a treated reaction mixture. The treated reaction mixture is maintained under depolymerizing conditions for a time period sufficient to permit the enzyme to depolymerize hybridized nucleic acid and release identifier nucleotides therefrom. The presence of released identifier nucleotides is analyzed to obtain an analytical output, the analytical output indicating the presence or absence of the nucleic acid target sequence. The analytical output may be obtained by various techniques as discussed above.
One method of the invention contemplates interrogating the presence or absence of a specific base in a nucleic acid target sequence in a sample to be assayed, and comprises the following steps.
A hybridization composition is formed by admixing a sample to be assayed with one or more nucleic acid probes under hybridizing conditions. The sample to be assayed may contain a nucleic acid target sequence to be interrogated. The nucleic acid target comprises at least one base whose presence or absence is to be identified. The hybridization composition includes at least one nucleic acid probe that is substantially complementary to the nucleic acid target sequence and comprises at least one predetermined nucleotide at an interrogation position, and an identifier nucleotide in the 3xe2x80x2-terminal region.
A treated sample is formed by maintaining the hybridization composition under hybridizing conditions for a time period sufficient for base pairing to occur when a probe nucleotide at an interrogation position is aligned with a base to be identified in the target sequence. A treated reaction mixture is formed by admixing the treated sample with an enzyme whose activity is to release one or more identifier nucleotides from the 3xe2x80x2-terminus of a hybridized nucleic acid probe to depolymerize the hybrid. The treated reaction mixture is maintained under depolymerizing conditions for a time period sufficient to permit the enzyme to depolymerize the hybridized nucleic acid and release an identifier nucleotide.
An analytical output is obtained by analyzing for the presence or absence of released identifier nucleotides. The analytical output indicates the presence or absence of the specific base or bases to be identified. The analytical output is obtained by various techniques, as discussed herein. Preferably, an identifier nucleotide is at the interrogation position.
In one aspect of a method of the invention, the nucleic acid target sequence is selected from the group consisting of deoxyribonucleic acid and ribonucleic acid.
A method that identifies the particular base present at an interrogation position, optionally comprises a first probe, a second probe, a third probe, and a fourth probe. An interrogation position of the first probe comprises a nucleic acid residue that is a deoxyadenosine or adenosine residue. An interrogation position of the second probe comprises a nucleic acid residue that is a deoxythymidine or uridine residue. An interrogation position of the third probe comprises a nucleic acid residue that is a deoxyguanosine or guanosine residue. An interrogation position of the fourth nucleic acid probe comprises a nucleic acid residue that is a deoxycytosine or cytosine residue.
In another aspect of the invention, the sample containing a plurality of target nucleic acid sequences is admixed with a plurality of the nucleic acid probes. Several analytical outputs can be obtained from such multiplexed assays. In a first embodiment, the analytical output obtained when at least one nucleic acid probes hybridizes with partial complementarity to one target nucleic acid sequence is greater than the analytical output when all of the nucleic acid probes hybridize with total complementarity to their respective nucleic acid target sequences. In a second embodiment, the analytical output obtained when at least one nucleic acid probe hybridizes with partial complementarity to one target nucleic acid sequence is less than the analytical output when all of the nucleic acid probes hybridize with total complementarity to their respective nucleic acid target sequences. In a third embodiment, the analytical output obtained when at least one nucleic acid probe hybridizes with total complementarity to one nucleic acid target sequence is greater than the analytical output when all of the nucleic acid probes hybridize with partial complementarity to their respective nucleic acid target sequences. In a fourth embodiment, the analytical output obtained when at least one nucleic acid probe hybridizes with total complementarity to one target nucleic acid sequence is less than the analytical output when all of the nucleic acid probes hybridize with partial complementarity to their respective nucleic acid target sequences. The depolymerizing enzymes are as described herein.
Yet another embodiment of the invention contemplates a method for determining the presence or absence of a first endogenous nucleic acid target in a nucleic acid sample that may contain that target or may contain a substantially identical second target. For example, the second target may have a base substitution, deletion or addition relative to the first nucleic acid target. This embodiment comprises the following steps.
A sample to be assayed is admixed with one or more nucleic acid probes under hybridizing conditions to form a hybridization composition. The first and second nucleic acid targets each comprise a region of sequence identity except for at least a single nucleotide at a predetermined position that differs between the targets. The nucleic acid probe is substantially complementary to the nucleic acid target region of sequence identity and comprises at least one nucleotide at an interrogation position. An interrogation position of the probe is aligned with the predetermined position of a target when a target and probe are hybridized. The probe also includes an identifier nucleotide in the 3xe2x80x2-terminal region.
The hybridization composition is maintained under hybridizing conditions for a time period sufficient to form a treated sample wherein the nucleotide at the interrogation position of the probe is aligned with the nucleotide at the predetermined position in the region of identity of the target.
A treated reaction mixture is formed by admixing the treated sample with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3xe2x80x2-terminus of a hybridized nucleic acid probe. The reaction mixture is maintained under depolymerization conditions for a time period sufficient to permit the enzyme to depolymerize the hybridized nucleic acid and release the identifier nucleotide.
An analytical output is obtained by analyzing for the presence or absence of released identifier nucleotides. The analytical output indicates the presence or absence of the nucleotide at the predetermined region, and; thereby, the presence or absence of a first nucleic acid target.
One aspect of the above method is comprised of a first probe and a second probe. The first probe comprises a nucleotide at an interrogation position that is complementary to a first nucleic acid target at a predetermined position. The second probe comprises a nucleotide at an interrogation position that is complementary to a second nucleic acid target at a predetermined position.
In one aspect of a process of the invention, the depolymerizing enzyme, whose activity is to release nucleotides, is a template-dependent polymerase, whose activity is to depolymerize hybridized nucleic acid whose 3xe2x80x2-terminal nucleotide is matched, in the 3xe2x80x2xe2x86x925xe2x80x2 direction in the presence of pyrophosphate ions to release one or more nucleotides. Thus, the enzyme""s activity is to depolymerize hybridized nucleic acid to release nucleotides under depolymerizing conditions. Preferably, this enzyme depolymerizes hybridized nucleic acids whose bases in the 3xe2x80x2-terminal region of the probe are matched with total complementarity to the corresponding bases of the nucleic acid target. The enzyme will continue to release properly paired bases from the 3xe2x80x2-terminus and will stop when the enzyme arrives at a base that is mismatched.
In an alternative aspect of the process (method), the depolymerizing enzyme, whose activity is to release nucleotides, exhibits a 3xe2x80x2xe2x86x925xe2x80x2 exonuclease activity in which hybridized nucleic acids having one or more mismatched bases at the 3xe2x80x2-terminus of the hybridized probe are depolymerized. Thus, the enzyme""s activity is to depolymerize hybridized nucleic acid to release nucleotides under depolymerizing conditions. In this embodiment, the hybrid may be separated from the free probe prior to enzyme treatment. In some embodiments, an excess of target may be used so that the concentration of free probe in the enzyme reaction is extremely low.
In still another alternative aspect of a process of the invention, the depolymerizing enzyme exhibits a 3xe2x80x2 to 5xe2x80x2 exonuclease activity on a double-stranded DNA substrate having one or more matched bases at the 3xe2x80x2 terminus of the hybrid. The enzyme""s activity is to depolymerize hybridized nucleic acid to release nucleotides containing a 5xe2x80x2 phosphate under depolymerizing conditions.
A further embodiment of the invention, such as is used for Single Tandem Repeat (STR) detection, contemplates a method for determining the number of known sequence repeats that are present in an endogenous nucleic acid target sequence in a nucleic acid sample. A method for determining the number of known sequence repeats comprises the following steps. A plurality of separate treated samples is provided. Each treated sample contains a nucleic acid target sequence hybridized with a nucleic acid probe. The nucleic acid target sequence contains a plurality of known sequence repeats and a downstream non-repeated region. Each nucleic acid probe contains a different number of complementary repeats of the known sequence, an identifier nucleotide in the 3xe2x80x2-terminal region and a 5xe2x80x2-terminal locker sequence. The 5xe2x80x2-terminal locker sequence is complementary to the downstream non-repeated region of the target and comprises 1 to about 20 nucleotides, preferably 5 to 20 nucleotides, most preferably 10 to 20 nucleotides. The various probes represent complements to possible alleles of the target nucleic acid. A treated depolymerization reaction mixture is formed by admixing each treated sample with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3xe2x80x2-terminus of a hybridized nucleic acid probe. The treated depolymerization reaction mixture is maintained under depolymerizing conditions for a time period sufficient to permit the enzyme to depolymerize the hybridized nucleic acid probe and release an identifier nucleotide. The samples are analyzed for the presence or absence of released identifier nucleotide to obtain an analytical output. The analytical output from the sample whose probe contained the same number of sequence repeats as present in the target nucleic acid is indicative of and determines the number of sequence repeats present in the nucleic acid target.
In one aspect of the method, the nucleic acid sample contains two nucleic acid targets representing alleles at a locus, and is homozygous with respect to the number of known sequence repeats of the two alleles. In an alternative method of the invention, the nucleic acid sample is heterozygous with respect to the two alleles at the locus. In another method of the invention, an identifier nucleotide is a nucleotide that is part of the region containing a repeated sequence. In an alternative method of the invention, an identifier nucleotide of the probe sequence is part of the region containing a non-repeating sequence that is complementary to that located in the target nucleic acid 5xe2x80x2 to the repeated known sequence. In this latter aspect of the method, the identifier nucleotide is present in a sequence containing 1 to about 20 nucleic acids that is complementary to a non-repeating sequence of the target nucleic acid located in the probe 3xe2x80x2 to the known sequence repeats. The repeated known sequence present in a nucleic acid target sequence typically has a length of 2 to about 24 bases per repeat.
A further embodiment of the invention contemplates a method using thermostable DNA polymerase as a depolymerizing enzyme for determining the presence or absence of at least one predetermined endogenous nucleic acid target sequence in a nucleic acid sample, and comprises the following steps.
A treated sample is provided that may contain a predetermined endogenous nucleic acid target sequence hybridized to a nucleic acid probe whose 3xe2x80x2-terminal region is complementary to the predetermined nucleic acid target sequence and includes an identifier nucleotide in the 3xe2x80x2-terminal region. A treated depolymerization reaction mixture is formed by admixing a treated sample with a depolymerizing amount of a enzyme whose activity is to release an identifier nucleotide from the 3xe2x80x2-terminus of a hybridized nucleic acid probe. In a preferred one-pot embodiment, the depolymerizing enzyme is thermostable and more preferably, the treated reaction mixture also contains (i) adenosine 5xe2x80x2 diphosphate, (ii) pyrophosphate, and (iii) a thermostable nucleoside diphosphate kinase (NDPK).
The treated sample is maintained under depolymerizing conditions at a temperature of about 4xc2x0 C. to about 90xc2x0 C., more preferably at a temperature of about 20xc2x0 C. to about 90xc2x0 C., and most preferably at a temperature of about 25xc2x0 C. to about 80xc2x0 C., for a time period sufficient to permit the depolymerizing enzyme to depolymerize the hybridized nucleic acid probe and release an identifier nucleotide as a nucleoside triphosphate. In preferred one-pot reactions, the time period is also sufficient to permit NDPK enzyme to transfer a phosphate from the released nucleoside triphosphate to added ADP, thereby forming ATP. The presence or absence of a nucleic acid target sequence is determined from the analytical output obtained using ATP. In a preferred method of the invention, analytical output is obtained by luminescence spectrometry.
In another aspect of the thermostable enzyme one-pot method for determining the presence or absence of a predetermined endogenous nucleic acid target sequence in a nucleic acid sample, the treated sample is formed by the following further steps. A hybridization composition is formed by admixing the sample to be assayed with one or more nucleic acid probes under hybridizing conditions. The 3xe2x80x2-terminal region of the nucleic acid probe (i) hybridizes with partial or total complementarity to a nucleic acid target sequence when that sequence is present in the sample, and (ii) includes an identifier nucleotide. A treated sample is formed by maintaining the hybridization composition under hybridizing conditions for a time period sufficient for the predetermined endogenous nucleic acid target sequence to hybridize with the nucleic acid probe.
Preferably, the depolymerizing enzyme is from a group of thermophilic DNA polymerases comprising Tne triple mutant DNA polymerase, Tne DNA polymerase, Taq DNA polymerase, Ath DNA polymerase, Tvu DNA polymerase, Bst DNA polymerase, and Tth DNA polymerase. The Tne triple mutant DNA polymerase is a preferred thermophilic enzyme and is discussed in greater detail hereinafter. In another aspect of the method, the NDPK is that encoded for by the thermophilic bacteria Pyrococcus furiosis (Pfu).
A still further method of the invention contemplates determining whether the presence or absence of a nucleic acid target sequence in a nucleic acid sample results from a locus that is homozygous or heterozygous for the two alleles at the locus. This method is comprised of the following steps. A plurality of separate treated samples is provided. Each sample may contain a nucleic acid target sequence hybridized with a nucleic acid probe. The nucleic acid target sequence consists of either a first allele, a second allele, or a mixture of first and second alleles of the nucleic acid target. The alleles differ in sequence at an interrogation position. The nucleic acid probe contains an identifier nucleotide in the 3xe2x80x2-terminal region that is aligned at an interrogation nucleotide position of the target sequence when the probe and target are hybridized.
A treated reaction mixture is formed by admixing each treated sample with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3xe2x80x2-terminus of a hybridized nucleic acid probe. The treated reaction mixture is maintained under depolymerizing conditions for a time period sufficient to permit the enzyme to depolymerize the hybridized nucleic acid probe and release an identifier nucleotide. The samples are analyzed for the presence or absence of released identifier nucleotides to obtain an analytical output. The analytical output is quantifiable and thus determines whether the sample is homozygous or heterozygous when compared to the analytical output of appropriate controls.
A multiplexed version of this embodiment is also contemplated, wherein probes for two or more alleles are provided in one reactionxe2x80x94each probe is distinguishable, but preferably each probe has the same length. Then, after hybridization, depolymerization, and analysis according to the invention, the relative analytical output for the various distinguishable identifier nucleotides or remaining probes will show whether the sample is homozygous or heterozygous and for which alleles. Another multiplexed version of this embodiment is contemplated, wherein probes for alleles at a plurality of loci are provided. Preferably, the different loci have substantially different target sequences. Probes for the various alleles at each locus are preferably of the same length. Each of the probes should be distinguishable either by analysis of the released identifier nucleotide or by analysis of the remaining probe after depolymerization.
Another embodiment of the invention contemplates a method for determining the loss of heterozygosity (LOH) of a locus of an allele that comprises the following steps.
A plurality of separate treated samples is provided, each sample containing a nucleic acid target sequence hybridized with a nucleic acid probe. The nucleic acid target sequence is that of a first allele or a mixture of the first allele and a second allele of the nucleic acid target, wherein the alleles differ in sequence. The nucleic acid probe contains a 3xe2x80x2-terminal region that hybridizes to a target sequence when the probe and target are hybridized.
Each treated sample is admixed with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3xe2x80x2-terminus of a hybridized nucleic acid probe to form a treated reaction mixture. The treated reaction mixture is maintained under depolymerizing conditions for a time period sufficient to depolymerize hybridized nucleic acid probe and release identifier nucleotides. The samples are then analyzed for the quantity of released identifier nucleotides to obtain an analytical output, the analytical output indicating whether the nucleic acid target sequence in a nucleic acid sample has lost heterozygosity at the locus of the allele.
In preferred LOH embodiments, the analytical output is obtained by luminescence spectroscopy, absorbance spectrometry, mass spectrometry or fluorescence spectroscopy. In another preferred embodiment, the released identifier nucleotide includes a fluorescent label. The identifier nucleotide is optionally fluorescently labeled after release from the hybrid.
It is contemplated that in the above analytical methods, either the released identifier nucleotide or the remainder of the probe can be evaluated to determine whether identifier nucleotide had been released, as described herein.
In another preferred LOH embodiment, the enzyme whose activity is to release nucleotides is a template-dependent polymerase that, in the presence of pyrophosphate ions, depolymerizes hybridized nucleic acids whose bases in the 3xe2x80x2-terminal region are completely complementary to bases of the nucleic acid target. The depolymerization proceeds from the 3xe2x80x2 terminal nucleotide of the probe and stops when it reaches a base that is not complementary to the corresponding target base.
In one aspect of the LOH embodiment, the quantity of the released identifier nucleotides for the first allele is substantially less than the quantity of the released identifier nucleotide for the first allele of a known heterozygous control sample, and the quantity of the released identifier nucleotides for the second allele is substantially similar to that of the released identifier nucleotide for the second allele of a known heterozygous control sample, indicating a loss of heterozygosity at the locus of the first allele.
In another aspect of the LOH embodiment, the quantity of the released identifier nucleotides for the second allele is substantially less than the quantity of the released identifier nucleotides for the second allele of a known heterozygous control sample, and the quantity of the released identifier nucleotides for the first allele is substantially similar to that of the released identifier nucleotide for the first allele of a known heterozygous control sample, indicating a loss of heterozygosity at the locus of the second allele. The known heterozygous control has analytical output for its treated sample indicating alleles one and two are present in the sample at about a 1:1 ratio. A sample with loss of heterozygosity has an analytical output for the treated samples indicating alleles one and two are present in the sample at a 1:0 or 0:1 ratio respectively when compared to the analytical output of a known heterozygous control sample.
A still further preferred embodiment of the invention contemplates a method for determining the presence of trisomy of an allele that comprises the following steps.
A plurality of separate treated samples is provided, wherein each sample contains a nucleic acid target sequence hybridized with a nucleic acid probe. The nucleic acid target sequence is that of a first allele, a second allele or a mixture of the first and second alleles of the nucleic acid target. The alleles differ in sequence at an interrogation position. The nucleic acid probe contains a 3xe2x80x2-terminal region that hybridizes to a region of the nucleic acid target sequence containing the interrogation nucleotide position when the probe and target are hybridized. The nucleic acid probe also contains an identifier nucleotide.
Each treated sample is admixed with a depolymerizing amount of an enzyme whose activity, under depolymerizing conditions, is to release one or more nucleotides from the 3xe2x80x2-terminus of a hybridized nucleic acid probe to form a treated reaction mixture. The treated reaction mixture is maintained for a time period sufficient to depolymerize hybridized nucleic acid probe and release identifier nucleotides. The samples are analyzed for released identifier nucleotides to obtain an analytical output, the magnitude of the analytical output relative to an analytical output of an appropriate control sample indicating whether a trisomy is present in the nucleic acid target sequence.
For trisomy analysis, preferably the analytical output is obtained by luminescence spectroscopy, absorbance spectrometry, fluorescence spectroscopy, or mass spectrometry. In one preferred embodiment, the released identifier nucleotide includes a fluorescent label. The identifier nucleotide is optionally fluorescently labeled after release from the hybrid.
In a preferred embodiment for trisomy analysis, the enzyme whose activity is to release nucleotides is a template-dependent polymerase, that, in the presence of pyrophosphate ions, depolymerizes hybridized nucleic acids whose bases in the 3xe2x80x2 terminal region are completely complementary to bases of said nucleic acid target.
In one embodiment, the quantity of released identifier nucleotides for the first allele is substantially greater than the quantity of the released identifier nucleotides of a control sample homozygous for the first allele, indicating that the nucleic acid target sequence has a trisomy. Preferably, the quantity of released identifier nucleotides is expressed as a ratio. For example, a normal heterozygote has about a 1:1 ratio of the analytical output for the two alleles. If the trisomy is homozygous for either allele, the ratio is about three times the value for that allele in a normal heterozygote that has none of the other allele. If the trisomy is heterozygous, then the ratio is about 2:1 of one allele to the other when compared to the analytical output of a control heterozygote.
A still further embodiment of the invention contemplates determining the presence or absence of a nucleic acid target sequence in a nucleic acid sample with a probe that is hybridized to the target and then modified to be able to form a hairpin structure. This embodiment comprises the following steps.
A treated sample is provided that contains a nucleic acid sample that may include a nucleic acid target sequence having an interrogation position hybridized with a nucleic acid probe. The probe is comprised of at least two sections. The first section contains the probe 3xe2x80x2-terminal about 10 to about 30 nucleotides. These nucleotides are complementary to the target strand sequence at positions beginning about 1 to about 30 nucleotides downstream of the interrogation position. The second section of the probe is located at the 5xe2x80x2-terminal region of the probe and contains about 10 to about 20 nucleotides of the target sequence. This sequence spans the region in the target from the nucleotide at or just upstream (5xe2x80x2) of the interrogation position, to the nucleotide just upstream to where the 3xe2x80x2-terminal nucleotide of the probe anneals to the target. An optional third section of the probe, from zero to about 50, and preferably about zero to about 20 nucleotides in length and comprising a sequence that does not hybridize with either the first or second section, is located between the first and second sections of the probe.
The probe of the treated sample is extended in a template-dependent manner, as by admixture with dNTPs and a template-dependent polymerase, at least through the interrogation position, thereby forming an extended probe/target hybrid. In a preferred embodiment, the length of the probe extension is limited by omission from the extension reaction of a dNTP complementary to a nucleotide of the target sequence that is present upstream of the interrogation position and absent between the nucleotide complementary to the 3xe2x80x2-end of the interrogation position.
The extended probe/target hybrid is separated from any unreacted dNTPs. The extended probe/target hybrid is denatured to separate the strands. The extended probe strand is permitted to form a hairpin structure.
A treated reaction mixture is formed by admixing the hairpin structure-containing composition with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3xe2x80x2-terminus of an extended probe hairpin structure. The reaction mixture is maintained under depolymerizing conditions for a time period sufficient for the depolymerizing enzyme to release 3xe2x80x2-terminus nucleotides, and then analyzed for the presence of released identifier nucleotides. The analytical output indicates the presence or absence of the nucleic acid target sequence.
A still further embodiment of the invention, termed REAPER(trademark), also utilizes hairpin structures. This method contemplates determining the presence or absence of a nucleic acid target sequence, or a specific base within the target sequence, in a nucleic acid sample, and comprises the following steps. A treated sample is provided that contains a nucleic acid sample that may include a nucleic acid target sequence hybridized with a first nucleic acid probe strand.
The hybrid is termed the first hybrid. The first probe is comprised of at least two sections. The first section contains the probe 3xe2x80x2-terminal about 10 to about 30 nucleotides that are complementary to the target nucleic acid sequence at a position beginning about 5 to about 30 nucleotides downstream of the target interrogation position. The second section of the first probe contains about 5 to about 30 nucleotides that are a repeat of the target sequence from the interrogation position to about 10 to about 30 nucleotides downstream of the interrogation position, and does not hybridize to the first section of the probe. An optional third section of the probe, located between the first and second sections of the probe, is zero to about 50, preferably up to about 20, nucleotides in length and comprises a sequence that does not hybridize to either the first or second section.
The first hybrid in the treated sample is extended at the 3xe2x80x2-end of the first probe, thereby extending the first probe past the interrogation position and forming an extended first hybrid whose sequence includes an interrogation position. The extended first hybrid is comprised of the original target nucleic acid and extended first probe. The extended first hybrid is then denatured in an aqueous composition to separate the two nucleic acid strands of the hybridized duplex and form an aqueous solution containing a separated target nucleic acid and a separated extended first probe.
A second probe, that is about 10 to about 2000, preferably about 10 to about 200, most preferably about 10 to about 30 nucleotides in length and is complementary to the extended first probe at a position beginning about 5 to about 2000, preferably about 5 to about 200, nucleotides downstream of the interrogation position in extended first probe, is annealed to the extended first probe, thereby forming the second hybrid. The second hybrid is extended at the 3xe2x80x2-end of the second probe until that extension reaches the 5xe2x80x2-end of the extended first probe, thereby forming a second extended hybrid whose 3xe2x80x2-region includes an identifier nucleotide. In preferred embodiments the extending polymerase for both extensions does not add a nucleotide to the 3xe2x80x2 end that does not have a corresponding complementary nucleotide in the template.
An aqueous composition of the extended second hybrid is denatured to separate the two nucleic acid strands. The aqueous composition so formed is cooled to form a xe2x80x9chairpin structurexe2x80x9d from the separated extended second probe when the target sequence is present in the original nucleic acid sample.
A treated reaction mixture is formed by admixing the hairpin structure-containing composition with a depolymerizing amount of an enzyme whose activity is to release one or more nucleotides from the 3xe2x80x2-terminus of a nucleic acid hybrid. The reaction mixture is maintained under depolymerizing conditions for a time period sufficient to release 3xe2x80x2-terminal region identifier nucleotides, and then analyzed for the presence of released identifier nucleotides. The analytical output indicates the presence or absence of the nucleic acid target sequence.
The present invention has many benefits and advantages, several of which are listed below.
One benefit of the invention is that, in some embodiments, nucleic acid hybrids can be detected with very high levels of sensitivity without the need for radiochemicals or electrophoresis.
An advantage of the invention is that the presence or absence of one or more target nucleic acid(s) can be detected reliably, reproducibly, and with great sensitivity.
A further benefit of the invention is that quantitative information can be obtained about the amount of a target nucleic acid sequence in a sample.
A further advantage of the invention is that very slight differences in nucleic acid sequence are detectable, including single nucleotide polymorphisms (SNPs).
Yet another benefit of the invention is that the presence or absence of a number of target nucleic acid sequences can be determined in the same assay.
Yet another advantage of the invention is that the presence or absence of a target nucleic acid can be determined with a small number of reagents and manipulations.
Another benefit of the invention is that the processes lend themselves to automation.
Still another benefit of the invention is its flexibility of use in many different types of applications and assays including, but not limited to, detection of mutations, translocations, and SNPs in nucleic acid (including those associated with genetic disease), determination of viral load, species identification, sample contamination, and analysis of forensic samples.
Still further benefits and advantages of the invention will become apparent from the specification and claims that follow.